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Protein kinase Cdelta stimulates proteasome-dependent degradation of C/EBPalpha during apoptosis induction of leukemic cells.

Zhao M, Duan XF, Zhao XY, Zhang B, Lu Y, Liu W, Cheng JK, Chen GQ - PLoS ONE (2009)

Bottom Line: More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment.Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT

Background: The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction.

Methodology/principal findings: Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.

Conclusions/significance: These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

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Related in: MedlinePlus

The proteolytically activated PKCδ is critical for the enhanced C/EBPα degradation during apoptosis.(A-B) After pretreatment for 2 hours in the presence or absence of 2 µM and 1 µM of rottlerin respectively for U937 and NB4 cells, U937 cells were treated with or without 50 nM of NSC606985 for an additional 36 hours or NB4 cells were treated with or without 25 nM of NSC606985 for an additional 18 hours. Then, the indicated proteins (top panel, A), relative CEBPA mRNA (bottom panel, A), and annexin-V+ cells% (B) were tested. (C-D) U937Flag-DN-PKCδ and U937vehicle cells were treated with 50 nM NSC606985 for 36 hours. Then, the indicated proteins (C) and annexin-V + cells%(D) were measured. (E) U937mock and U937PKCδ-CF cells were incubated for the indicated days after tetracycline withdrawal, and the indicated proteins (top panel) and relative CEBPA mRNA (bottom panel) were determined. (F) U937mock and U937 PKCδ -CF cells were incubated in medium with (+) or without (−) tetracycline for 4 days, and annexin-V + cells% were measured on flow cytometry. Here, Δcaspase-3, ΔPKCδ and ΔPARP indicate activated fragments of caspase-3, cleaved 41kDa catalytic fragments of PKCδ and cleaved fragments of PARP, respectively. In panel C, Flag-DN-PKCδ was detected by anti-Flag antibody, and the symbol * indicated a non-corresponding band. In panel A/E, the columns represent means of change folds of CEBPA mRNA against untreated cells, with the bar as S.D. of three independent experiments with triplicates each. In panel B, D and F, the values represent annexin V+ cells with and without PI staining, as expressed by mean±S.D. of three independent experiments each with triplicates, and the symbol * represents P<0.01 compared with NSC606985 treatment alone (B), U937vehicle cells (D) or U937mock cells with the corresponding treatment.
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pone-0006552-g003: The proteolytically activated PKCδ is critical for the enhanced C/EBPα degradation during apoptosis.(A-B) After pretreatment for 2 hours in the presence or absence of 2 µM and 1 µM of rottlerin respectively for U937 and NB4 cells, U937 cells were treated with or without 50 nM of NSC606985 for an additional 36 hours or NB4 cells were treated with or without 25 nM of NSC606985 for an additional 18 hours. Then, the indicated proteins (top panel, A), relative CEBPA mRNA (bottom panel, A), and annexin-V+ cells% (B) were tested. (C-D) U937Flag-DN-PKCδ and U937vehicle cells were treated with 50 nM NSC606985 for 36 hours. Then, the indicated proteins (C) and annexin-V + cells%(D) were measured. (E) U937mock and U937PKCδ-CF cells were incubated for the indicated days after tetracycline withdrawal, and the indicated proteins (top panel) and relative CEBPA mRNA (bottom panel) were determined. (F) U937mock and U937 PKCδ -CF cells were incubated in medium with (+) or without (−) tetracycline for 4 days, and annexin-V + cells% were measured on flow cytometry. Here, Δcaspase-3, ΔPKCδ and ΔPARP indicate activated fragments of caspase-3, cleaved 41kDa catalytic fragments of PKCδ and cleaved fragments of PARP, respectively. In panel C, Flag-DN-PKCδ was detected by anti-Flag antibody, and the symbol * indicated a non-corresponding band. In panel A/E, the columns represent means of change folds of CEBPA mRNA against untreated cells, with the bar as S.D. of three independent experiments with triplicates each. In panel B, D and F, the values represent annexin V+ cells with and without PI staining, as expressed by mean±S.D. of three independent experiments each with triplicates, and the symbol * represents P<0.01 compared with NSC606985 treatment alone (B), U937vehicle cells (D) or U937mock cells with the corresponding treatment.

Mentions: It has been known that PKCδ, a novel member of the PKC family, is implicated as an important regulator of apoptotic responses, especially in DNA-damaging agents-induced apoptosis [14], [17], [18]. The proteolytic activation of PKCδ, which results in the generation of an active kinase domain [19], occurs in response to a variety of stimuli including DNA-damaging agents and exerts a crucial role in NSC606985/etoposide-induced apoptosis [9], [14], [20]. In line with our previous reports [14], [21], NSC606985/etoposide induced a proteolytic cleavage of PKCδ into a 41kDa catalytic fragment (Figure 1A-C) that persistently activated the kinase [20]. Of note, the PKCδ antibody also detected a strong band between the full-length PKCδ and the catalytic fragment (CF) of PKCδ, which also disappeared when cells are treated with apoptotic stimuli. What this band actually corresponds to remained to be explored. Also, it should be pointed out that the increase of truncated PKCδ had a good correlation with the decrease of full-length PKCδ after 24 hours of NSC606985 treatment in NB4 cells, but such correlation was not so remarkable in NSC606985 or etoposide-treated U937 cells. We extrapolated that this might be due to higher sensitivity of NB4 cells than U937 cells to etoposide and especially NSC606985-induced apoptosis (Figure S1/S2). The proteolytic activation of PKCδ was also present in UV, doxorubicin and As2O3-induced apoptosis (Figure 1D). Considering that the proteolytic activated PKCδ protein could target some proteins for their degradation [22] and the PKCδ activation also occurred before the reduced C/EBPα protein according to time-course analysis (Figure 1A/C and ref [14]), we extrapolated that the PKCδ activation was related to the degradation of C/EBPα protein. Hence, U937 and NB4 cells were pretreated with the specific PKCδ inhibitor rottlerin, which significantly inhibited NSC606985-induced proteolytic activation of PKCδ (Figure 3A). Likewise, it also antagonized activation of caspase-3, cleavage of its substrate poly-ADP ribose polymerase (PARP) (Figure 3A) and apoptosis (Figure 3B) induced by NSC606985. In parallel, rottlerin also partially restored C/EBPα protein but not its mRNA level upon NSC606985 treatment in both cells (Figure 3A).


Protein kinase Cdelta stimulates proteasome-dependent degradation of C/EBPalpha during apoptosis induction of leukemic cells.

Zhao M, Duan XF, Zhao XY, Zhang B, Lu Y, Liu W, Cheng JK, Chen GQ - PLoS ONE (2009)

The proteolytically activated PKCδ is critical for the enhanced C/EBPα degradation during apoptosis.(A-B) After pretreatment for 2 hours in the presence or absence of 2 µM and 1 µM of rottlerin respectively for U937 and NB4 cells, U937 cells were treated with or without 50 nM of NSC606985 for an additional 36 hours or NB4 cells were treated with or without 25 nM of NSC606985 for an additional 18 hours. Then, the indicated proteins (top panel, A), relative CEBPA mRNA (bottom panel, A), and annexin-V+ cells% (B) were tested. (C-D) U937Flag-DN-PKCδ and U937vehicle cells were treated with 50 nM NSC606985 for 36 hours. Then, the indicated proteins (C) and annexin-V + cells%(D) were measured. (E) U937mock and U937PKCδ-CF cells were incubated for the indicated days after tetracycline withdrawal, and the indicated proteins (top panel) and relative CEBPA mRNA (bottom panel) were determined. (F) U937mock and U937 PKCδ -CF cells were incubated in medium with (+) or without (−) tetracycline for 4 days, and annexin-V + cells% were measured on flow cytometry. Here, Δcaspase-3, ΔPKCδ and ΔPARP indicate activated fragments of caspase-3, cleaved 41kDa catalytic fragments of PKCδ and cleaved fragments of PARP, respectively. In panel C, Flag-DN-PKCδ was detected by anti-Flag antibody, and the symbol * indicated a non-corresponding band. In panel A/E, the columns represent means of change folds of CEBPA mRNA against untreated cells, with the bar as S.D. of three independent experiments with triplicates each. In panel B, D and F, the values represent annexin V+ cells with and without PI staining, as expressed by mean±S.D. of three independent experiments each with triplicates, and the symbol * represents P<0.01 compared with NSC606985 treatment alone (B), U937vehicle cells (D) or U937mock cells with the corresponding treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2719015&req=5

pone-0006552-g003: The proteolytically activated PKCδ is critical for the enhanced C/EBPα degradation during apoptosis.(A-B) After pretreatment for 2 hours in the presence or absence of 2 µM and 1 µM of rottlerin respectively for U937 and NB4 cells, U937 cells were treated with or without 50 nM of NSC606985 for an additional 36 hours or NB4 cells were treated with or without 25 nM of NSC606985 for an additional 18 hours. Then, the indicated proteins (top panel, A), relative CEBPA mRNA (bottom panel, A), and annexin-V+ cells% (B) were tested. (C-D) U937Flag-DN-PKCδ and U937vehicle cells were treated with 50 nM NSC606985 for 36 hours. Then, the indicated proteins (C) and annexin-V + cells%(D) were measured. (E) U937mock and U937PKCδ-CF cells were incubated for the indicated days after tetracycline withdrawal, and the indicated proteins (top panel) and relative CEBPA mRNA (bottom panel) were determined. (F) U937mock and U937 PKCδ -CF cells were incubated in medium with (+) or without (−) tetracycline for 4 days, and annexin-V + cells% were measured on flow cytometry. Here, Δcaspase-3, ΔPKCδ and ΔPARP indicate activated fragments of caspase-3, cleaved 41kDa catalytic fragments of PKCδ and cleaved fragments of PARP, respectively. In panel C, Flag-DN-PKCδ was detected by anti-Flag antibody, and the symbol * indicated a non-corresponding band. In panel A/E, the columns represent means of change folds of CEBPA mRNA against untreated cells, with the bar as S.D. of three independent experiments with triplicates each. In panel B, D and F, the values represent annexin V+ cells with and without PI staining, as expressed by mean±S.D. of three independent experiments each with triplicates, and the symbol * represents P<0.01 compared with NSC606985 treatment alone (B), U937vehicle cells (D) or U937mock cells with the corresponding treatment.
Mentions: It has been known that PKCδ, a novel member of the PKC family, is implicated as an important regulator of apoptotic responses, especially in DNA-damaging agents-induced apoptosis [14], [17], [18]. The proteolytic activation of PKCδ, which results in the generation of an active kinase domain [19], occurs in response to a variety of stimuli including DNA-damaging agents and exerts a crucial role in NSC606985/etoposide-induced apoptosis [9], [14], [20]. In line with our previous reports [14], [21], NSC606985/etoposide induced a proteolytic cleavage of PKCδ into a 41kDa catalytic fragment (Figure 1A-C) that persistently activated the kinase [20]. Of note, the PKCδ antibody also detected a strong band between the full-length PKCδ and the catalytic fragment (CF) of PKCδ, which also disappeared when cells are treated with apoptotic stimuli. What this band actually corresponds to remained to be explored. Also, it should be pointed out that the increase of truncated PKCδ had a good correlation with the decrease of full-length PKCδ after 24 hours of NSC606985 treatment in NB4 cells, but such correlation was not so remarkable in NSC606985 or etoposide-treated U937 cells. We extrapolated that this might be due to higher sensitivity of NB4 cells than U937 cells to etoposide and especially NSC606985-induced apoptosis (Figure S1/S2). The proteolytic activation of PKCδ was also present in UV, doxorubicin and As2O3-induced apoptosis (Figure 1D). Considering that the proteolytic activated PKCδ protein could target some proteins for their degradation [22] and the PKCδ activation also occurred before the reduced C/EBPα protein according to time-course analysis (Figure 1A/C and ref [14]), we extrapolated that the PKCδ activation was related to the degradation of C/EBPα protein. Hence, U937 and NB4 cells were pretreated with the specific PKCδ inhibitor rottlerin, which significantly inhibited NSC606985-induced proteolytic activation of PKCδ (Figure 3A). Likewise, it also antagonized activation of caspase-3, cleavage of its substrate poly-ADP ribose polymerase (PARP) (Figure 3A) and apoptosis (Figure 3B) induced by NSC606985. In parallel, rottlerin also partially restored C/EBPα protein but not its mRNA level upon NSC606985 treatment in both cells (Figure 3A).

Bottom Line: More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment.Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT

Background: The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction.

Methodology/principal findings: Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.

Conclusions/significance: These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

Show MeSH
Related in: MedlinePlus